The invention relates to a transgenic non-human mammal, to a method for producing the latter, to the utilization thereof, to a cell tissue therefrom, to a method for producing such cell tissue, to the utilization thereof, to a recombinant DNA expression vector and to the utilization of such vector.xe2x80x94The term non-human mammal refers to taxonomically higher units than animal species. Transgenic animals are organisms carrying an additional gene not originating from their species, that is a foreign gene in their genome. For the purpose of the invention in particular such transgenic animals are meant that have the foreign gene also in the germ cells, that is which hand on the foreign gene vertically, i.e. from generation to generation. If a special transgenic animal has been created, further corresponding transgenic animals may be obtained by breeding. The transgenic animals are known in the art in various embodiments, and various methods for producing transgenic animals are also known. As an example only, reference is made to document R. Jaenisch, Science, Vol. 240, 10, 1988, page 1468 ff., and the documents cited therein. The term cell tissue comprises complete organs or parts of organs of an animal, however also specific cell lines that can be isolated and cultivated therefrom, i.e. Increased in number.
A recombinant DNA expression vector is an instrument for producing a transgenic animal carrying, among other features, the foreign DNA to be integrated in the cells of the animal.
The general technological background of the special transgenic animal provided by the invention is the following. Cancer, in particular lung cancer, is one of the most widespread diseases of mankind, and has up to now therapy predictions offering little success only. In the framework of the development of better therapies for cancer diseases it is, among other conditions, required and also legally laid down, for ethical reasons, to perform pre-clinical examinations in animals models with possible active substances obtained from basic research or by screening tests. In the case of examinations of prospective active substances for cancer therapies, it is therefore required to provide animals or (animal) cell tissues having the respective cancer diseases to be investigated, in order that the physiological effects, possibly also side effects, of the active substances can be tested in a qualitative and quantitative manner.
Cancer diseases are caused in many cases by the effects of so-called oncoproteins. These are proteins that have different structures compared to corresponding proteins in a healthy organism. These oncoproteins are capable, through not yet fully understood processes, to transform normal cells into uncontrollably proliferating cells, i.e. cancer cells. The formation of oncoproteins in an organism is in turn caused by so-called oncogenes, i.e. genes coding for the oncoprotein. Oncogenes may be introduced into a cell by viruses, may however also be formed by way of mutation of (certain) xe2x80x9chealthyxe2x80x9d genes, the proto-oncogenes. Such mutations can for instance take place by translocation (displacement) of a gene responsible for the production of a protein within the genome, by point mutations (replacement of a base and/or individual bases in the DNA of a gene responsible for the production of a protein by different base, with the consequence of the formation of a protein of modified amino acid sequence, the oncogene), by deletion (removal of one or more bases) or also by mutations in the region of a so-called promoter applicable for the respective gene. As a promoter is designated a DNA region of a gene by means of which the transcription (of the DNA code into a corresponding RNA) and thus finally also the expression (formation) of the protein correlated with the gene can be controlled. In a natural manner, a specific promoter is usually assigned to each gene, this promoter being arranged ahead of the latter in the genome. Ahead means that the promoter in the DNA sequence has a certain distance to the starting point of a transcription. For initiating a transcription, it is then also required that so-called transcription factors (often specific for the cell type) are taken up by the promoter.
In particular in connection with lung cancer, the so-called Raf proto-oncogenes play a special role. These genes are highly conservative with regard to evolution, and code kinases specific for serine/threonine of the cytoplasm playing in turn a role in the mitogenic signal transduction. Known in the art are for instance the genes A, B and c-Raf-1. For a survey, reference is made to documents U. R. Rapp et al., The Oncogene Handbook, Elsevier Science Publishers, Netherlands, page 115-154, 1988, and U. R. Rapp, Oncogene, 6, 495, 1991. To the family of the Raf genes belongs, among others, the c-Raf-1 gene expressing the c-Raf-1 kinase ubiquitarily in an organism. The c-Raf-1 gene comprises three conserved regions, i.e. these regions are in accordance with corresponding regions of other Raf genes of the family. The region CR1 is a regulatory domain around a cys finder consensus sequence, the region CR2 is a region having a high content of serine or threonine, and CR3 is the kinase domain. With regard to further detailed information, reference is made to document U.S. Pat. No. 5,618,670. From this document are also known (partial) sequences of the natural form of the CR3 region of the c-Raf-1 gene of mice and (partial) sequences of various point mutations thereof. From document U.S. Pat. No. 5,156,841 are known plasmids and eucariotic expression vectors containing A-Raf and v-Raf oncogenes, however in different connections, namely the genic production of Raf oncoproteins for immunological investigations.
The invention is based on the technical problem to provide non-human mammals in sufficiently high number and with a pathology being uniform and reproducible with regard to tumor formation, for the purpose of pre-clinic examinations of prospective anti-cancer substances or therapies.
For achieving this object, the invention teaches a transgenic non-human mammal whose cells express a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or a protein coded by a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene. xe2x80x94The term constitutively active means, in the context of the invention, that the protein per se is always active, i.e. the physiological effect of the protein is always obtained even without the condition of further reaction cascades in a cell or an organism. In contrast thereto, the activation of the not constitutively active Raf-1 protein requires for instance the bonding of the Ras protein with the Raf-1 protein. The term constitutively active therefore refers, for the purpose of the invention, only to the protein or the corresponding gene code and not to the gene itself or the gene activation. The reference to the Raf-1 gene means Raf-1 genes or variants thereof existing in any organism, at least however such Raf-1 genes existing in mammals. By the fact that the mammal is a transgenic animal having the mentioned features, identical animals, with regard to the pathology of the tumors induced by the expression of the mutant, can be obtained in any number by way of the natural reproduction from a transgenic base animal. Thereby pre-clinic examinations of active substances or therapies can be performed with the required reproducibility and the required statistical significance, and that also with defined control groups.
For performing pre-clinic examinations of active substances and therapies against lung cancer it is recommended that the expression of the protein coded by the constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or by a corresponding allele or a derivative of the A, B or c-Raf-1 gene takes place in lung cells, since then the animals develop reproducible lung tumors.
In structural regard, a mammal as described above is characterized by that it contains foreign DNA with constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or with a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene. Alleles or derivatives are variants in a DNA sequence virtually not affecting the basic function of the respective gene.
Advantageously, the foreign DNA in addition contains a promoter for the surfactant protein C, preferably for the human surfactant protein C, and this promoter is arranged in the foreign DNA with the proviso that by the promoter the transcription of the mutant of the kinase-domain of the c-Raf-1 gene or of a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene is controlled. In other words, the promoter being in a natural manner arranged ahead of the gene coding the surfactant protein C, is arranged instead, according to the invention, at a suitable position of the mutant or of the gene. The precise arrangement of promoter and mutant or gene with regard to each other is usual knowledge of the man skilled in the art. If the exact positioning of the special promoter ahead of the gene utilized according to the invention cannot be derived from basic considerations, simple tests with different variants of positioning can however be performed, in order to determine a suitable position. The number of variants in question, under consideration of the general technical knowledge, is however very limited. The surfactant protein C plays a role for the surfactant factor reducing the alveolar surface tension between the lung epithelium and air and thus preventing that the alveoli will collapse during breathing-out and that the epithelia with stick together. By application of the promoter for the surfactant protein C it is achieved that only the transcription factors specifically or with increased frequency occurring in the lung and inter-reacting with this promoter can so to speak switch on the mutant, with the result that lung tumors will be formed at high selectivity and reproducibility.
In a preferred further embodiment of the invention, the foreign DNA in addition contains DNA of the SV40 virus, SC means Simian Virus. This comprises the polyadenylation sequence and intron/exon regions of the SV40 virus being known in the art (see description of the following FIG. 1). The integration of this SV40 DNA causes an increase of the translation efficiency of the polyadenylated mRNA. In detail it is preferred that the mammal comes from the group of rodents.
A transgenic non-human mammal as described above is obtainable by the following steps: a) integration of the cDNA sequence of a constitutively active oncogenic mutant of the kinase-domain of the c-RAF-1 gene or of a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene in an expression vector, b) insertion of the transgenic vector obtained in step a), preferably after linearization, in pronuclei of fertilized oocytes from a non-human mammal, c) implantation of the oocytes obtained in step b) in brood animals of the same species as the donor species of the oocytes and delivery of descendant animals from the oocytes, d) genotypization and selection of the descendant animals obtained in step c) with the proviso that cells of the selected mammals express a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or a protein coded by a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene. Genotypization can be obtained by means of methods well known to the man skilled in the art, for instance by tail biopsy by means of PCR (polymerase chain reaction, a method for the in-vitro amplification of a defined DNA fragment) and Southern Blot (a method for the analysis of DNA fragments in DNA preparations), with those mammals being selected whose cells can be proven by the examinations of the genotypization to contain the foreign DNA according to the invention. Advantageously, the expression vector used is step a) contains a promoter for the surfactant protein C, preferably a promoter for the human surfactant protein C. This promoter is arranged in the foreign DNA with the proviso that by the promoter the transcription of the mutant of the kinase-domain of the Raf-1 gene or of a corresponding normal allele or derivative of the A, B or c-Raf-1 gene is controlled. In detail, the foreign DNA may comprise for instance either the healthy (FIG. 1) or a constitutively active oncogenic mutant of the Raf-1 gene with a sequence according to one of FIG. 1, however with deletion as xcex94Raf (26-302). In place of a deletion, point mutations of the sequence shown in FIG. 1 can also be used. Such point mutations can for instance specifically be caused by administration of 1-ethyl-1-nitrosourea (ENU) to animals having the healthy sequence, thereby such mutants being accessible in a simple manner.
The invention also relates to a method for producing a transgenic non-human mammal. Non-human mammals according to the invention are used for pre-clinic examinations of the effectiveness of substances directed against lung carcinomas and/or therapeutical methods directed against lung carcinomas, in particular for the pre-clinic examination of the effectiveness of substances inhibiting Raf-kinase. Such substances completely inhibit or reduce the activity of Raf-kinases, thereby possibly a means for the deactivation particularly of Raf-oncoproteins and therefore for the proliferation inhibition of tumor cells being found. Another advantageous utilization of a non-human mammal according to the invention is the investigation of the pathogenesis of lung tumors, thereby a better understanding of the disease per se being possible.
The invention however also relates to cell tissues, in particular lung tissues, from a transgenic non-human mammal, which cell tissue has a higher probability of tumor formation, preferably of lung tumors, to a method for the production thereof and to the utilization thereof. With regard to the cell tissue or also specific cell lines comprised therein, isolated and possibly cultivated therefrom, all general explanations given above apply in corresponding manner.
Finally the invention also comprises a recombinant DNA expression vector containing A) the DNA sequence of a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or of a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene, B) a promoter domain for the surfactant protein C, by means of which the transcription of the DNA sequence defined in A) is controllable, C) as an option the DNA sequence of the SV40 virus. By means of such a vector the transgenic mammals according to the invention and possibly also the cell tissues therefrom can be produced. As an example, the DNA sequence defined in A) comprises a sequence according to FIG. 4 or to the end of the specification, respectively, or a sequence xcex94Raf (26-302) derived therefrom, and/or the promoter domain defined in B) is a promoter domain for the human surfactant protein C. The reproduced sequence is that of human-c-Raf-1. Instead, also the c-Raf-1 sequences of the mouse, wild type or mutated, according to document U.S. Pat. No. 5,618,670 can be used. Other sequences, even from other organisms, are also possible, as far as they are basically a Raf-1 sequence.
Subject matter of the invention is further a screening method with utilization of the transgenic non-human mammals according to the invention or of cell tissues therefrom, a group of prospective active substances against cancer, in particular lunger cancer, being administered to the animals, and an evaluation of the effects of each individual prospective active substance with regard to proliferation inhibition, oncoprotein inhibition or the like is performed. The invention further comprises active substances that can be detected by such a screening method as being sufficiently effective.
Plasmids with transgenic vectors according to the invention (active oncogenic mutant of the kinase-domain of the human c-Raf-1 gene, i.e. plasmid SPC-xcex94Raf (26-302) and a transgenic vector with normal human c-Raf-1 gene, i.e. plasmid SPC-Raf-1) were registered at DSMZxe2x80x94Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, D-3812 Braunscheweig. The registration number for the plasmid SPC-xcex94Raf (26-302) is 11849. Thus the invention also relates to transgenic vectors as registered, as well as to transgenic animals or cells or cell tissues to be produced therefrom, and to the utilizations described above of such animals or cells or cell tissues.